A number of methods are known in the art that use an oligonucleotide that exhibits a proto-promoter sequence to synthesize first-strand cDNA and then double-stranded cDNA that contains an RNA polymerase promoter (e.g., in order to amplify RNA molecules in a sample for gene expression analysis and other purposes). A “proto-promoter sequence” is a single-stranded DNA or RNA sequence region which, in double-stranded DNA form, is capable of mediating RNA transcription by serving as an RNA polymerase promoter.
One of the most common methods for evaluating the expression of mRNA transcripts in a small biological sample comprises: synthesizing first-strand cDNA using an oligo(dT) promoter primer; synthesizing second-strand cDNA using the fragments of the mRNA or a first-strand cDNA hairpin as a primer and the first-strand cDNA as a template; and transcribing the double-stranded cDNA containing an RNA polymerase promoter using RNA polymerase, as described by Van Gelder et al. in U.S. Pat. Nos. 5,545,522; 5,716,785; and 5,891,636. Thus, the second-strand cDNA is the template strand and the RNA synthesized is complementary to (or “anti-sense” to) the mRNA in the sample. The single-stranded promoter sequence exhibited by the promoter primer of Van Gelder et al., which is one strand of a functional double-stranded promoter, is referred to herein as an “anti-sense promoter sequence” and the corresponding promoter primer is referred to herein as an “anti-sense promoter primer.” Similarly, the promoter sequence of a double-stranded promoter that is operably joined to the template strand is referred to herein as a “sense promoter sequence.” For example, but without limiting the invention with respect to the promoter sequence or the respective RNA polymerase used for transcription, whereas one T7 RNA polymerase anti-sense promoter sequence and +1 base exhibited by a promoter primer in U.S. Pat. Nos. 5,545,522; 5,716,785; and 5,891,636 is:
(5′ TAATACGACTCACTATAG, 3; SEQ ID NO: 6)the corresponding T7 RNA polymerase sense promoter sequence and +1 base is:
(5′ CTATAGTGAGTCGTATTA, 3′. SEQ ID NO: 7)
A number of methods are known in the art for using RNA in a sample as a template and an oligonucleotide with a proto-promoter sequence to synthesize double-stranded cDNA and then labeled anti-sense RNA (“aRNA”) (e.g., for gene expression profiling and other applications), including, for example, methods described in: Murakawa et al., DNA 7:287-295, 1988; Phillips and Eberwine, Methods in Enzymol. Suppl. 10:283-288, 1996; Ginsberg et al., Ann. Neurol. 45:174-181, 1999; Ginsberg et al., Ann. Neurol. 48:77-87, 2000; VanGelder et al., Proc. Natl. Acad. Sci. USA 87:1663-1667, 1990; Eberwine et al., Proc. Natl. Acad. Sci. USA 89:3010-3014, 1992; U.S. Pat. Nos. 5,021,335; 5,130,238; 5,168,038; 5,399,491; 5,437,990; 5,545,522; 5,514,545; 5,665,545; 5,716,785; 5,891,636; 5,958,688; 6,291,170; PCT Patent Applications WO 00/75356 and WO 02/065093; and U.S. Patent Application Nos. 20020127592; Kamme: 20030175714; and Scheinert: 20060035226.
Other methods use in vitro transcription as part of a process for amplifying and detecting one or more nucleic acid sequences, including, for example, methods described in U.S. Pat. Nos. 5,194,370; 5,409,818; 5,466,586; 5,554,517; 6,063,603; 6,090,591; 6,100,024; 6,410,276; Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173, 1989; Fahy et al, In: PCR Methods and Applications, pp. 25-33, 1991; PCT Patent Application Nos. WO 89/06700 and WO 91/18155; and European Patent Application Nos. 0427073 A2 and 0427074 A2.
Although much less used, methods for synthesizing sense RNA molecules corresponding to RNA in a sample are also known in the art. For example, in U.S. Pat. No. 5,169,766, Schuster and Berninger disclosed a method for using a proto-promoter-containing nucleic acid molecule having a blocked 3′-terminus to add DNA that exhibited a sense promoter sequence to the 3′-termini of first-strand cDNA molecules. In U.S. Pat. Nos. 5,962,271 and 5,962,272, Chenchik et al. disclosed a method for using a template switching oligonucleotide to add an arbitrary sequence to the 3′-termini of first-strand cDNA molecules, which method has been coupled to the use of a PCR primer that exhibits an anti-sense-promoter sequence in its 5′-portion and a sequence that is complementary to the arbitrary sequence in its 3′-terminal portion to make double-stranded cDNA that is transcribed with an RNA polymerase to make multiple copies of sense RNA (Harris, J et al., Biochimica Biophysica Acta—General Subjects 1724: 127-136, 2005). In U.S. Patent Application No. 20030073112, Zhang et al. disclose use of a second-strand cDNA synthesis primer that is promoter primer with a random 3′-terminal sequence for synthesis of cDNA that can be transcribed. In U.S. Patent Application No. 20030186237 and PCT Patent Application No. WO 02/065093, Ginsberg al. disclosed a method to make sense RNA molecules by first using a method similar to Chenchik et al. to add a sequence tag, and then to generate double-stranded cDNA with an RNA polymerase promoter. In U.S. Patent Application No. 20040171041, Dahl et al. disclosed methods for joining DNA that exhibits a sense promoter sequence for an RNA polymerase promoter to the 3′-termini of first-strand cDNA molecules. In U.S. Patent Application No. 20050153333 and PCT Patent Application No. WO 2007062495, Sooknanan disclosed methods for selective terminal tagging of nucleic acids, which can be used for joining an RNA polymerase promoter for synthesis of sense RNA molecules. In U.S. Patent Application Nos. 20060281153; 20070048741; 20070105124; and 20080020431, Getts et al. disclosed methods to synthesize sense RNA after making cDNA molecules with an RNA polymerase promoter using terminal transferase and a single-stranded promoter template.
Although several methods have been disclosed for amplifying sense RNA molecules that correspond to RNA in a sample, none of these methods is widely used in the art. This is unfortunate because methods that synthesize multiple copies of sense RNA molecules corresponding to RNA molecules of interest in a sample provide several advantages over methods that synthesize anti-sense RNA molecules. For example, since the sense RNA molecules synthesized using some of these methods have an RNA sequence tag joined to their 5′-termini, they can be used for subsequent rounds of synthesis of more first-strand cDNA molecules that have a DNA sequence tag joined to their 3′-termini and more sense RNA molecules that have the RNA sequence tag joined to their 5′-termini. The RNA sequence tag thereby fixes the length of the cDNA product and of the sense RNA product synthesized in the subsequent rounds. Thus, although the methods that use an anti-sense promoter primer (e.g., the methods described in U.S. Pat. Nos. 5,545,522; 5,716,785; and 5,891,636) result in anti-sense RNA products that have a 3′-bias (i.e., the anti-sense RNA molecules synthesized using the method exhibit sequences derived from the 3′-portions of the RNA molecules of interest to a greater extent than they exhibit sequences derived from the 5′-portions of the RNA molecules of interest), and which, moreover, become shorter and more 3′-biased during each round of amplification, the methods that synthesize sense RNA have less 3′-bias and, due to the presence of the DNA and RNA sequence tags, the lengths of the products are generally maintained during each round of amplification. Sense RNA molecules are also advantageous over anti-sense RNA molecules because they exhibit substantially the same sequences as the RNA molecules of interest in the sample. Thus, for example, sense RNA molecules synthesized using mRNA molecules of interest can be used for in vitro or in vivo translation of proteins that exhibit substantially the same amino acid sequences as those which are present in the sample.
One reason the methods for synthesis of sense RNA are not widely used in the art in spite of their advantages is that they generally result in synthesis of significant quantities of non-specific background RNA that is not related to the RNA molecules of interest in the sample. In general, excess primers and primer-induced artifacts (e.g., primers which are tagged with a sequence tag and with an RNA polymerase promoter) are key sources of the non-specific background. Thus, the methods in the art typically require time-consuming and tedious steps, such as use of mini-columns, in order to try to remove the sources of the background. Often, significant background remains even after these purification steps because not all of the nucleic acid molecules that contribute to the background are removed. At the same time, the purification steps can result in significant losses of nucleic acid molecules that are derived from the RNA molecules of interest, which can potentially decrease the sensitivity, or even affect the interpretation of the results obtained.
What is needed in the art are methods for synthesis of first-strand cDNA molecules, double-stranded cDNA molecules, and sense RNA molecules that provide the benefits of using the respective sequence tags (e.g., less 3′-sequence bias and fixed product lengths during multiple rounds of synthesis), but which do not result in synthesis of significant quantities of non-specific background RNA that is not related to the RNA molecules of interest in the sample. What is needed are methods for synthesizing first-strand cDNA molecules, double-stranded cDNA molecules, and sense RNA molecules wherein tedious and time-consuming mini-column or other purification steps, particularly purification steps that result in losses of nucleic acid molecules derived from the RNA molecules of interest, are minimized or avoided. What is needed are methods that are more efficient for obtaining first-strand cDNA molecules that have a DNA sequence tag joined to their 3′-termini and sense RNA molecules that have an RNA sequence tag joined to their 5′-termini. What is needed are methods to obtain such amplified sense RNA molecules that exhibit substantially the same sequences and that are present in substantially the same abundances as the RNA molecules of interest in the sample, including mRNA or miRNA or ncRNA molecules of interest from small biological samples, for applications such as expression analysis, and for preparing sense RNA molecules for transfection into a eukaryotic or prokaryotic cell to study or cause a biological effect, for in vivo translation into protein in prokaryotic or eukaryotic cells, or for in vitro translation in a cell-free system. What is needed are methods that enable generation of complete libraries of cDNA molecules and complete libraries of sense RNA molecules, which represent all of the RNA molecules of interest in a sample, including even a sample consisting of approximately 1000 to 10,000, 100 to 1000, 10 to 100, or even 1 to 10 cells.